Codon Bias as a means to fine-tune Gene Expression

Review article by Quax et al., 2015 Published in Molecular cell

General principle of protein expression

A ribosome and translation

Orientation of Codon and Anticodon

Genetic Code degeneracy

• 20 amino acids are encoded by multiple synonymous codons (61 codons and 3 stop codons).

• Overabundance of codons allows many amino acids to be encoded by more than one codon.

• For this reason genetic code is said to be degenerated.

Synonymous and non synonymous mutations

• Miscopied DNA nucleotide change only one nucleotide.

• Results in coding for same amino acid

• Insertion or deletion of single nucleotide.

• Cause frame shift mutation.

Codon bias• A non random distribution of synonymous codons in genes

of different organisms.

• Each organism prefer a different set of codons over other.

General codon bias variants• Codon Adaptation Index (CAI):

The CAI for a specific gene can be determined by comparing its codon usage frequency with reference set of highly expressed genes.

• Wobbling and tRNA modification:

Translation of multiple synonymous codons by single tRNA .

It can be G-U, I-U, I-A or I-C

Translation of mRNA with a wobbling codon anticodon base pairing

Correlation of codon bias with tRNA pool

• tRNA adaptation index (tAI):

The copy number of tRNA genes are assumed to be correlated with tRNA abundance in cells.

The organisms with larger genomes have higher tRNA gene redundancy which would decrease selection for specific codons.

Codon frequency are correlated with total supply of tRNA.

• Normalized translation efficiency (nTE):

More frequently used codon recognized by more abundant tRNA species, the codon will compete for this tRNA with other codons

Cont’d…. nTE provides the information for supply as well as demand

rates of tRNAs.

Highly expressed proteins are encoded by genes that contain relatively high proportions of codons recognized by abundant, charged tRNAs with kinetically efficient codon-anticodon base pairing.

Types of Codon BiasSynonymous Codon Co-occurrence

Non synonymous Pair Base

• Synonymous codons of a coding sequence are not randomly distributed rather they are biased to cluster those codons that are recognized by same tRNA.

• The codon resides under selective constraint

• Nucleotides neighboring a particular codon are distributed in non random manner.

Is Translation efficiency correlated with codon bias?

• The translation efficiency and resultant protein is determined by both translation initiation and elongation rates.

Translation initiation rate controls how often the transcript is translated.

Translation elongation rate controls the speed of translation process.

Influence of coding sequence on translation initiation

• Translation starts when the ribosome is sequestered on mRNA

In prokaryotes the shine-Dalgarno sequence in mRNA binds with the anti shine-dalgarno sequence in 16S rRNA gene.

In eukaryotes the Kozak sequence around the start codon is involved in interaction with pre initiation complex for translation.

The strength of mRNA folding around the initiation sequence and start codon can influence translation initiation efficiency.

Is Translation elongation rate controlled by codon bias?

• High translation initiation and elongation rate is required for optimal expression.

• Low translational initiation and elongation rate will give low expression.

• More frequently used codons recognized by abundant tRNA results in faster translation and higher translation efficiency.

• Ribosome density profiling is also used to analyze the distribution of ribosomes on mRNA.

Intragenic fluctuations in translation efficiency

Intergenic fluctuations in translation efficiency

Intragenic codon landscape and expression

• Besides codon frequency bias on genome level, local codon bias within gene is also observed.

• Many genes possess locally biased distributions of rare and frequent codons resulting in Codon Landscape.

• Variable local translations can:

Regulate even distribution of ribosomes on mRNA

Tuning of protein co-translational folding process

Facilitate protein translocation across membranes

Cont’d…

• Rare codon ramps to reduce ribosome jamming:

It is immediately downstream of the start codon (30-50 rare codons).

It is usually translated with high efficiency to support fast release of initiator tRNA met then slows down until it reaches elongation.

Slow start of elongation process facilitate evenly spacing of ribosomes on mRNA to reduce jamming during further elongation of highly expressed proteins.

Codon landscape for protein translation across membrane

• Rare codon clusters were identified in genes for membrane and secretory proteins.

• In yeast 35-40 codons downstream of binding sites for the signal recognition particle is present.

Codon landscape and co-translational protein folding

• Clusters of rare codons may helps in co-translational protein folding

• nTE metric suggested frequent codons are depleted from the regions that encode coils in protein structures.

Rare codons are present in alpha helix at position 1 & 4 whereas frequent codons are present at position 2 &3 suggesting complex co-translational folding.

Beta sheets are encoded by frequent codons suggesting a correlation between codon bias and co-translational folding.

Intergenic codon bias and differential expression

• Difference in codon bias is not only between regions within individual genes but also between sets of genes either clustered in operons or scattered in a genome.

Starvation condition and codon bias:• Rare codon clusters encodes amino acid biosynthesis pathway.

• Amino acids starvation results in selective charging .

• Charging levels of some tRNA will be low & some will remain high.

• Rare Codons read highly charged tRNA , used for efficient translation of genes essential during starvation.

Cont’d…Cell cycle, differentiation and stress regulation by codon usage:• In humans and other vertebrates tRNA conc. differ in differentiating cell types.• Cyanobacterium uses codons to adjust protein production during fluctuating environmental conditions.• tRNA modifications can alter codon-anticodon binding affinities and translations of certain condons can be favored.

Selection pressure on codon usage

• Codon usage is biased in majority of living organism

• Two explanations on the evolution of this bias are:

Non-random mutation:

• Codon bias is related to non random mutation caused by GC content.

Selection for codon bias:

• Codon usage bias related to translational efficiencies.

• Codon bias must be under selective pressure during evolution and mutations rate cannot explain alone the various observations.

Applying codon bias as a means to improve protein

• Codon usage bias has been studied to be used as a strategy in BIOTECHNOLOGY to optimize gene expression for improving protein production and yield.

Expressing additional tRNA genes in the production host:

• Heterologous expression of gene

• Consisting of additional copy number of tRNA genes

• Enhances tRNA levels

• Examples: E.coli Rosetta (pRARE)

• E.coli BL21-CodonPlus (pRIL)

Cont’d… Designing codon optimized genes:• DNA synthesis companies offer codon optimization services

• Optimization is done by maximizing gene’s CAI that matches with host’s expression

• Sequencing features that are taken into account are:

GC content

Avoidance of repeats and RNase recognition sites

Transcriptional terminator site

Shine-Dalgarno sequences

mRNA folding sequence sites

Challenges ahead: unraveling codon bias and other factors influencing expression

• Comparative analysis of coding sequences and protein productions that influence translation process.

• Improvements, integration of experimental approaches and statistical analysis.

• Analysis of experimental RNA sequencing data that determines mRNA and tRNA abundance, ribosome density profiling and proteomics.

• Designing synthetic gene variants will be more efficient than generating and testing random reported gene variants.

Further improving synthetic gene design

• Synthetic genes should contain as much as possible frequent codons in order to achieve high protein production in heterologous production system.

• These features have hardly used in synthetic gene design therefore, deserve more attention in future attempts.

• Codon optimization can be obtained by analysis of aminoacyl tRNA abundance ensuring balanced tRNa supply needed for protein production.

• nTE for predicting optimal codon bias should be considered.

Applying codon bias as a tool in synthetic biology

• Improved gene designs may lead to

Synthetic gene circuits, biosynthesis pathways, new genomes

• Engineering at genome level

• Replacement of rare codons by frequent codons on genome wide scale level

• Rare codons reassigned to encode non natural amino acids

Thank You..